Method for producing car gene-introduced nk cells and use thereof

ABSTRACT

The present invention relates to a method for producing induced natural killer (iNK) cells, into which a chimeric antigen receptor (CAR) gene encoding a CAR is introduced, iNK cells produced by the method, and a cell therapy composition and a pharmaceutical composition for preventing or treating cancer, comprising the iNK cells.The method according to the present invention has the effects of producing the iNK cells, into which a CAR gene is introduced, with high efficiency through direct reprogramming from isolated cells without limiting an initial cell, and directly producing the same without a differentiation process, thereby simplifying the production process and reducing costs and time. The method according to the present invention has the effect of producing excellent NK cells having enhanced safety by directly producing NK cells from human somatic cells that are easy to obtain, without passing through induced pluripotent stem cells produced through conventional reprogramming technology. In addition, the iNK cells, into which a CAR gene is introduced, produced by the method, have an excellent cancer cell killing ability, and thus can be effectively utilized as a cell therapy composition or a pharmaceutical composition for preventing or treating cancer.

TECHNICAL FIELD

The present invention relates to a method for producing induced natural killer (iNK) cells into which a chimeric antigen receptor (CAR) gene encoding a CAR is introduced, iNK cells produced by the method, and a cell therapy composition and a pharmaceutical composition for preventing or treating cancer, including the iNK cells.

BACKGROUND ART

NK (natural killer) cells are key innate immune cells that perform the body's primary defense (innate immunity) function by immediately recognizing and eliminating infections of viruses, bacteria and parasites, and abnormal autologous cells (especially cancer cells). Unlike T cells, which recognize target cells by expressing antigen-specific receptors, NK cells perceive abnormal changes in target cells (especially cancer cells), such as balance of inhibitory or activating receptors, i.e., killer immunoglobulin receptors (KIR), natural cytotoxicity receptors (NCR), DNAM-1 (DNAX accessory molecule-1), and NKG2D (NK group 2 member D, and loss of surface MHC (major histocompatibility complex) class I antigen, without antigen specificity and human leukocyte antigen (HLA) matching, and have contact-dependent cytotoxicity.

Specifically, NK cells can kill cancer cells by directly mediating target cancer cell apoptosis through secretion of cytokines, such as perforin (Prf1), granzyme B (GzmB), interferon-γ (IFN-γ), interleukin (tumor necrosis factor-α; TNF-α), etc., and activation of apoptosis-inducing receptors (Fas, tumor necrosis factor-related apoptosis-inducing ligand; TRAIL). In addition, it is known that NK cells that express Fc receptor-FcRγIIIa (CD16) can effectively eliminate cancer cells directly or indirectly by inducing acquired immunity as well as innate immunity, such as mediating antibody-dependent cellular cytotoxicity (ADCC). In particular, unlike T cells, NK cells do not cause side effects such as graft-versus-host disease (GVHD), and thus have gained attention as a safe cell source that can be used for the development of not only autologous but also allogeneic anticancer immune cell therapeutics.

Currently, human NK cells are produced by isolating and proliferating a small amount of cells present in the human body [peripheral blood, bone marrow, umbilical cord blood, etc.] through a primary culture method, or can be obtained multidimensionally from stem cells having the ability to differentiate into NK cells [hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs)] through differentiation induction culture.

Recently, it has been demonstrated that T cells (CAR-T cells), which are produced by introducing a cancer cell target chimeric antigen receptor (CAR) gene as a method for promoting specificity and activation for target cancer cells and consequently enhancing the efficacy of anticancer treatment, have an enhanced anticancer effect in the treatment of blood cancer, and thus have received much interest in the development of CAR-immune cell therapeutics. However, cytokine release syndrome (CRS) induced by interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 factors secreted by CAR-T cells is recognized as a serious problem that needs to be solved. Further, CAR-T cells are known to inhibit cancer cell cytotoxicity by the immune checkpoint protein PD (programmed death ligand)-1, etc. in the tumor microenvironment (TME) of solid tumors, and accordingly, there is a need for technology development to improve the efficacy of solid cancer treatments.

However, since NK cells secrete IL-3 and granulocyte-macrophage colony-stimulating factor, they are less likely to cause CRS [K. Rezvani, R. Rouce, E. Liu et al., Engineering Natural Killer Cells for Cancer Immunotherapy, Mol. Ther., 25 (2017), pp. 1769-1781]. Additionally, the level of PD-1 secreted by NK cells is substantially low, and almost no immunosuppression is induced, and also, it is known that NK cells enable migration of dendritic cells to tumors, thereby enhancing anti-PD-1 immunotherapy [K. C. Barry, J. Hsu, M. L. Broz, et al. A Natural Killer-dendritic Cell Axis Defines Checkpoint Therapy-responsive Tumor Microenvironments, Nat. Med., 24 (2018), pp. 1178-1191], and thus, the excellence of CAR-NK development has been demonstrated. Further, it was confirmed from the results of preclinical and clinical trials for CAR-NK cell therapy that NK cells can effectively remove blood cancer and solid cancer cells [E. L. Siegler, Y. Zhu, P. Wang, et al. Off-the-shelf CAR-NK Cells for Cancer Immunotherapy, Cell Stem Cell, 23 (2018), pp. 160-161], and thus have been recognized to have the potential to be developed as anticancer immune cell therapeutics in a wide range, including for solid cancers.

As a method for producing CAR-NK cells, a method for introducing a CAR gene specific for a cancer antigen into a single NK cell line (NK-92, iPSC, etc.) that is relatively easy to culture and proliferate, and isolating and amplifying CRA-expressing NK cells is mainly used. The most used primary cultured NK-92 cells are cells derived from patients with malignant non-Hodgkin's lymphoma and have the potential to induce secondary tumorigenesis and EB (Epstein-Barr) virus sensitivity after injection. Thus, in order to ensure safety, a lethal irradiation process is essential before clinical application, but this process shortens the in vivo survival time of CAR-NK-92 cells, and consequently, the anticancer effect is reduced. The method of producing NK cells by introducing a CAR gene into induced pluripotent stem cells (iPSCs) and inducing differentiation essentially requires not only the CAR-iPSCs process, and isolation and amplification process, but also a complex differentiation process that involves much time and cost, and carries the possibility of tumor formation, and thus, it is essential to develop a technology that can secure and maintain safety.

Recently, a technology for directly producing high-value-added functional human tissue-specific target cells having characteristics of a different lineage from initial human somatic cells, which are relatively easy to obtain using reprogramming technology of somatic cells, is rapidly developing. However, there has been no report on a technology for directly producing CAR-NK cells without passing through iPSCs by somatic cell reprogramming.

DISCLOSURE Technical Problem

The present inventors have made extensive efforts to develop a method for efficiently producing CAR-NK cells, and as a result, they have developed a CAR-NK cell-specific reprogramming medium and reprogramming culture conditions, and confirmed that CAR-NK cells can be produced from human somatic cells by a method that does not require a differentiation process without limiting the initial cell resources, and accordingly, the produced CAR-NK cells exhibit an excellent cancer cell killing ability and thus can be applied to the prevention or treatment of cancer, thereby completing the present invention.

Technical Solution

It is one object of the present invention to provide a method for producing CAR-iNK (induced natural killer) cells, including: culturing isolated cells, into which a reprogramming factor and a CAR (chimeric antigen receptor) gene are introduced, sequentially in a) a first medium containing growth factors, cytokines, and a GSK3β (glycogen synthase kinase 3 beta) inhibitor; b) a second medium containing growth factors, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist; and c) a third medium containing cytokines, a GSK3β inhibitor, and an AHR antagonist, and thereby directly reprogramming into NK cells.

It is another object of the present invention to provide CAR-iNK cells produced according to the method.

It is still another object of the present invention to provide a cell therapy composition for preventing or treating cancer, including the CAR-iNK cells produced according to the method above, as an active ingredient.

It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, including the CAR-iNK cells produced according to the method above, as an active ingredient.

Advantageous Effects

The method according to the present invention can produce iNK cells, into which a CAR gene is introduced, from the isolated cells without a differentiation process through direct reprogramming, thereby producing CAR-NK cells with an enhanced cancer cell killing function with high efficiency without limitation of the initial cells, and has an effect of reducing cost and time by simplifying the production process. Since NK cells can be produced without passing through induced pluripotent stem cells produced through a conventional reprogramming technique, there is an excellent NK cell production effect which is enhanced and distinguished in terms of safety. In addition, the CAR gene-introduced iNK cells produced by the above method have an excellent cancer cell killing ability, and thus can be effectively utilized as a cell therapy composition or a pharmaceutical composition for preventing or treating cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a diagram showing the constitutional domains of four CAR genes.

FIG. 2 is a schematic diagram showing a method for producing CAR-iNK by direct somatic cell reprogramming and CAR gene introduction.

FIG. 3 is a result confirming CD19-CAR-iNK cells produced from PBMC.

FIG. 4 is a result confirming MSLN-CAR-iNK cells produced from PBMC.

FIG. 5 is a result confirming HER2-CAR-iNK cells produced from PBMC.

FIG. 6 (A) shows a result confirming the CD19-CAR-iNK cells produced according to the introduction period of CAR-expressing lentivirus (day 0, day 14, day 24), and (B) shows a result confirming the insertion of the CD19-CAR gene in the iNK cells produced from PBMC.

FIG. 7 is a result confirming the CAR-iNK production efficiency according to the composition or incubation time of the CAR-iNK first medium and third medium.

FIG. 8 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.

FIG. 9 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.

FIG. 10 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced MSLN-CAR-iNK cells and cancer cells.

FIG. 11 is a result confirming the frequency of CD107a-expressing cells during co-culture of the produced HER2-CAR-iNK cells and cancer cells.

FIG. 12 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.

FIG. 13 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced CD19-CAR-iNK cells and cancer cells.

FIG. 14 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced MSLN-CAR-iNK cells and cancer cells.

FIG. 15 is a result confirming the frequency of IFN-gamma-expressing cells during co-culture of the produced HER2-CAR-iNK cells and cancer cells.

FIG. 16 is a result confirming the cancer cell killing ability of the produced CD19-CAR-iNK cells.

FIG. 17 is a result confirming the cancer cell killing ability of the produced MSLN-CAR-iNK cells.

FIG. 18 is a result confirming the cancer cell killing ability of the produced HER2-CAR-iNK cells.

FIG. 19 is a result confirming the tumor growth inhibitory effect of MSLN-CAR-iNK cells in a pancreatic cancer cell xenograft mouse animal model.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will be described in detail below. Meanwhile, each description and embodiment disclosed herein can be applied to other descriptions and embodiments, respectively. That is, all combinations of various elements disclosed herein fall within the scope of the present invention. Further, the scope of the present invention is not limited by the specific description described below.

In order to achieve the above objects, one aspect of the present invention provides a method for producing CAR-iNK (induced natural killer) cells, including: culturing isolated cells, into which a reprogramming factor and a CAR (chimeric antigen receptor) gene are introduced, sequentially in a) a first medium containing growth factors, cytokines, and a GSK3β (glycogen synthase kinase 3 beta) inhibitor; b) a second medium containing growth factors, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist; and c) a third medium containing cytokines, a GSK3β inhibitor, and an AHR antagonist, and thereby directly reprogramming into NK cells.

As used herein, the “NK (natural killer) cell” is a key innate immune cell that performs the body's first defense (innate immunity) function to eliminate infections of viruses, bacteria and parasites, and abnormal autologous cells (cancer cells, etc.). NK cells have been proven to be effective in preventing the occurrence, proliferation, metastasis, and recurrence of various types of cancers including not only blood cancer but solid cancers, and thus have received attention as a useful cellular resource for anticancer therapeutics and relapse inhibitors. Efforts to develop NK cells isolated and cultured from the human body and NK cells differentiated from stem cells as anticancer immune cell therapeutics are rapidly increasing, but low NK production efficiency and anticancer treatment efficacy are still recognized as problems to be overcome.

As a method to promote the specificity and activation for cancer cells, NK (CAR-NK) cells expressing a chimeric antigen receptor (CAR) specific to various cancer antigens were produced, and accordingly, studies are being actively conducted to investigate the enhancement of cancer cell killing ability and its therapeutic efficacy. As a method for producing the CAR-NK cells, a method of introducing cells into a single NK cell line (NK-92, iPSCs, etc.) that is relatively easy to culture and proliferate is mainly used, but low production efficiency, a complicated process, and safety issues (possibility of tumor formation, etc.) have been recognized as problems to be overcome.

Therefore, the present inventors have attempted to produce CAR-NK cells using a method that is applicable to various human somatic cells that are easy to collect under ex vivo culture conditions and does not require a differentiation process, and a result, they have identified for the first time a method for directly inducing and producing CAR-NK cells from isolated human somatic cells through direct reprogramming.

As used herein, the term “CAR-iNK (CAR-induced natural killer) cell” refers to an NK (natural killer) cell which is induced through direct reprogramming according to the method of the present invention and into which a CAR gene is introduced.

As used herein, the term “reprogramming” refers to a method of converting a lineage into a target cell having completely different characteristics by controlling the global gene expression pattern of a specific cell. Reprogramming may include dedifferentiation of cells, direct reprogramming or direct conversion, or direct trans-differentiation, but is not limited thereto. In the present invention, the reprogramming may be performed by introducing a vector containing a foreign gene or DNA into a cell. As used herein, the term “transformation” refers to the change of a cell to a different state, and the term “differentiation” refers to a phenomenon in which daughter cells produced by cell division acquire a function different from that of the original parent cell, and as used herein, the “conversion” and “differentiation” can be used interchangeably with “induction”.

As used herein, the term “direct reprogramming” refers to a method of inducing direct conversion to a target cell by culturing a specific cell in a reprogramming medium. In order to produce NK cells, which are target cells, using conventional reprogramming techniques, 1) induced pluripotent stem cells were produced from isolated somatic cells; 2) hematopoietic stem (progenitor) cells, the intermediate, were subjected to primary differentiation and production from induced pluripotent stem cells; 3) and subsequently, NK cells, which are target cells, were subjected to secondary differentiation and production from differentiated stem (progenitor) cells. As described above, the conventional technique has the disadvantages of low production efficiency and high time and cost consumption, because it has to sequentially go through a complex culture process. In addition, since NK cells are produced via induced pluripotent stem cells with pluripotency, the remaining undifferentiated cells have the potential to form tumors, and thus safety is an important issue to be verified. In contrast, the present invention produces NK cells, which are the target cells, directly from isolated somatic cells through direct reprogramming, thereby providing reduced production time and cost, and excellent efficiency and safety, and thus can be distinguished from the prior art and can provide an alternative that can overcome the problems. The direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct cross-differentiation, cross-differentiation, etc., and as used herein, the direct reprogramming may mean direct dedifferentiation or cross-differentiation into NK cells from isolated somatic cells, but is not limited thereto.

As used herein, the term “differentiated cells” refers to a state in which cells with specialized structures or functions, that is, cells, tissues, etc. of living organisms, have changed into a form and function suitable for performing the role assigned thereto. For example, the differentiated cells broadly refer to ectodermal, mesodermal, and endodermal cells derived from pluripotent stem cells such as embryonic stem cells, and narrowly to red blood cells, white blood cells, platelets, etc. derived from hematopoietic stem cells.

As used herein, the “lineage-conversion cell” is a cell which is converted to a cell type with different lineage characteristics due to the change in the intrinsic lineage characteristics of the cell embryologically or artificially (e.g., reprogramming, etc.), thereby having the characteristics of a cell type that are completely different from the characteristics of the cell type before conversion. In the present invention, the lineage-conversion cell may be a target cell. For example, non-NK lymphocyte cells in peripheral blood mononuclear cells may be converted to NK cells in a reprogramming medium, but are not limited thereto.

In the present invention, the method may be performed by culturing isolated cells, into which a reprogramming factor and a CAR gene are introduced, sequentially in a) a first medium containing growth factors, cytokines, and a GSK3β (glycogen synthase kinase 3 beta) inhibitor; b) a second medium containing growth factors, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist; and c) a third medium containing cytokines, a GSK3β inhibitor, and an AHR antagonist, and thereby directly reprogramming into NK cells.

As used herein, the term “isolated cells” are not particularly limited, but specifically refer to cells whose lineage has already been specified, such as germ cells, somatic cells, or progenitor cells. The “somatic cells” refer to all cells in which differentiation constituting animals and plants has been completed except for germ cells. The “progenitor cells” refer to a mother cell which does not express a differentiated character, but has a differentiation fate, if it has been found that a cell corresponding to its progeny expresses a certain differentiation character. For example, as for the nerve cells (neurons), nerve fibroblasts (neuronal stem cells) correspond to the precursor cells, and as for the myotube, myoblasts correspond to the precursor cell.

The isolated cells may be cells derived from a human, but are not limited thereto, and cells derived from various individuals may also fall within the scope of the present invention. In addition, the isolated cells of the present invention may include both in vivo or ex vivo cells. Specifically, the isolated cells may be somatic cells, and more specifically, somatic cells other than NK cells, but are not limited thereto.

As used herein, the term “reprogramming factor” refers to a gene (or polynucleotide) that can be introduced into a cell to induce reprogramming, or a protein encoded therefrom. The reprogramming factor may vary depending on the target cell to be obtained through reprogramming, and the type of cell before reprogramming. For example, when isolated somatic cells are to be induced into NK cells, the reprogramming factor introduced into the isolated somatic cells may include any one or more selected from the group consisting of Lin28, Asc11, Pitx3, Nurr1, Lmx1a, Nanog, Oct4, Oct3, Sox2, Klf4, Myc, and a combination thereof, and specifically Oct4, Sox2, Klf4, and Myc, but is not limited thereto, and may include any factor known in the art as long as it is a reprogramming factor that can induce the isolated somatic cells into NK cells. The reprogramming using the reprogramming factor is the induction of conversion to a target cell by controlling the entire gene expression pattern of the cell, and the cell may be reprogrammed into a target cell having a gene expression pattern of a desired type of cell by introducing the reprogramming factor into the cell and culturing the same for a certain period of time.

As used herein, the “introduction of reprogramming factor” may be a method of administering a reprogramming factor to a culture solution of cells; a method of directly injecting a reprogramming factors into cells; a method of increasing the expression level of a reprogramming factor present in a cell; a method of transforming a cell with an expression vector containing a gene encoding a reprogramming factor; a method of modifying a gene sequence to increase the expression of a gene encoding a reprogramming factor; a method of introducing an exogenously expressed gene encoding a reprogramming factor; a method of treating a substance having an effect of inducing expression of the reprogramming factor; and a method of increasing the expression level of a reprogramming factor in a cell through a combination thereof, but is not limited thereto as long as it can increase the expression level of the reprogramming factor. In particular, the introduction of reprogramming factor may be inducing expression of a reprogramming factor depending on a desired time and conditions. Specifically, the method of introducing a reprogramming factor into a cell may be a method of administering a reprogramming factor to a cell culture solution, or a method of transforming a cell with an expression vector containing a gene encoding a reprogramming factor, but this not limited.

The method of directly injecting a reprogramming factor into a cell may be performed by selecting any method known in the art, but is not limited thereto, and may be performed by appropriately selecting from the methods using microinjection, electroporation, particle bombardment, direct muscle injection, an insulator, and a transposon.

As used herein, the term “vector” refers to a DNA construct containing the nucleotide sequence of a suitable regulatory sequence and the target protein or polypeptide so as to be able to express the target protein or polypeptide in a suitable host cell. The regulatory sequence may include a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer, etc. The vector of the present invention may include a signal sequence or a leader sequence for membrane targeting or secretion, in addition to the regulatory sequence, and can be prepared in various ways depending on the desired purpose. The promoter of the vector may be constitutive or inducible. Further, the vector may include a selective marker for selecting a host cell containing the vector, and in the case of a replicable vector, may include a replication origin. Once transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may integrate into the genome thereof.

The vector used in the present invention is not particularly limited as long as it is able to replicate in the host cell, and any vector known in the art may be used. Examples of the vector conventionally used may include natural or recombinant virus vector, episomal vector, plasmid vector, cosmid vector, etc.

Specifically, the virus vector may include vectors derived from retrovirus such as Sendai virus, lentivirus, HIV (human immunodeficiency virus, MLV (murine leukemia virus), ASLV (avian sarcoma/leukosis), SNV (spleen necrosis virus), RSV (Rous sarcoma virus), MMTV (mouse mammary tumor virus), etc., adenovirus, adeno-associated virus, herpes simplex virus, and more specifically, it may be an RNA-based virus vector, but is not limited thereto.

The episomal vector is a non-viral non-insertable vector, and is known to have a property of expressing a gene included in the vector without being inserted into a chromosome. Accordingly, the cell containing the episomal vector may include both cases in which the episomal vector is inserted into the genome or is present in a cell without being inserted into the genome.

As used above, the term “operably linked” refers to a functional linkage between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein so as to perform a general function. The operative linkage with the recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation are carried out using enzymes generally known in the art.

As used herein, the term “CAR gene” refers to a gene encoding a chimeric antigen receptor consisting of an extracellular domain, a transmembrane domain, and an intracellular domain including genes encoding the extracellular domain, the transmembrane domain, and the intracellular domain including an antibody domain (scFv). For the purpose of the present invention, the CAR gene may be any one or more selected from the group consisting of a CD19-CAR1 gene or CD19-CAR2 gene including CD19 scFv, a MSLN-CAR gene including MSLN (mesothelin) scFv, and a HER2-CAR gene including HER2 (human epidermal growth factor receptor 2) scFv, but is not limited thereto.

It is known that the CAR target factors for solid tumors include EGFRvIII (Morgan R A, Hum Gene Ther. 2012; 23:1043-1053), MUC-1 (Wilkie S, J Immunol. 2008; 180:4901-4909), MAGE (Willemsen R A, Gene Ther. 2001; 8:1601-1608), CEA (Emtage P C, Clin Cancer Res. 2008; 14:8112-8122), PSMA, GD2, CA125, Her2 and MSLN, FAP, VEGFR (Kakarla S, Cancer J. 2014; 20:151-155), etc.

Additionally, the CD19 is the cluster of differentiation (CD) assigned with number 19 for identifying cell surface molecules according to the immunophenotype, and the CD19 refers to a marker of B lymphocytes. The CD19 is known to be expressed in most B-cell malignant cancer cells and thus provides an ideal target for these carcinomas.

Specifically, the CAR gene may be any one or more selected from the group consisting of:

i) a CAR gene (CD19-CAR1 gene) including CD8 leader, CD19 scFv, CD8 hinge, CD8 transmembrane domain, and Fc-γ receptor;

ii) a CAR gene (CD19-CAR2 gene) including CD8 leader, CD19 scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3ζ, and IRES (internal ribosome entry site);

iii) a CAR gene (MSLN-CAR gene) including CD8 leader, MSLN (mesothelin) scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3ζ, and IRE; and

iv) a CAR gene (HER2-CAR gene) including CD8 leader, HER2 (human epidermal growth factor receptor 2) scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3ζ, and IRES, but is not limited thereto.

The CD8 leader may include a nucleotide sequence of SEQ ID NO: 1, CD19 scFv may include a nucleotide sequence of SEQ ID NO: 2, MSLN scFv may include a nucleotide sequence of SEQ ID NO: 3, HER2 scFv may include a nucleotide sequence of SEQ ID NO: 4, CD8 hinge may include a nucleotide sequence of SEQ ID NO: 5, CD8 transmembrane domain may include a nucleotide sequence of SEQ ID NO: 6, Fc-γ receptor may include a nucleotide sequence of SEQ ID NO: 7, CD28 intracellular domain may include a nucleotide sequence of SEQ ID NO: 8, CD3ζ may include a nucleotide sequence of SEQ ID NO: 9, and the IRES inserted to clone the CAR gene into a vector constituting a double cistron may include a nucleotide sequence of SEQ ID NO: 10, but is not limited thereto.

The CAR gene may further include GFP (green fluorescent protein), but is not limited thereto.

The GFP may include a nucleotide sequence of SEQ ID NO: 11, but is not limited thereto.

The nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11 can be confirmed from NCBI Genbank, a known database.

In the present invention, the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11 may include a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1 to SEQ ID NO: 11. Additionally, it is apparent that a nucleotide sequence in which a part of the sequence is deleted, modified, substituted, or added may be included within the scope of the present invention as long as the nucleotide sequence has such homology or identity and exhibits a function corresponding to the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 11.

As used herein, the term “homology and identity” refers to a degree of relatedness between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity may often be used interchangeably with each other.

The sequence homology or identity of conserved polynucleotides or polypeptide may be determined by standard alignment algorithms and can be used with a default gap penalty established by the program being used. Substantially, homologous or identical sequences are generally expected to hybridize to all or at least about 50%, 60%, 70%, 80%, or 90% or more of the entire length of the sequences under moderate or high stringent conditions. Polynucleotides that contain degenerate codons instead of codons in hybridizing polynucleotides are also considered.

The homology or identity of the polypeptide or polynucleotide sequences may be determined by, for example, BLAST algorithm by literature [see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873(1993)], or FASTA by Pearson (see Methods Enzymol., 183, 63, 1990). Based on the algorithm BLAST, a program referred to as BLASTN or BLASTX has been developed (see: http://www.ncbi.nlm.nih.gov). Further, whether any two amino add or polynucleotide sequences have a homology, similarity, or identity with each other may be identified by comparing the sequences in a Southern hybridization experiment under stringent conditions as defined, and appropriate hybridization conditions defined are within the skill of the art, and may be determined by a method well known to those skilled in the art (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology).

In the present invention, the CAR gene may be introduced into the cell in the same manner as the above-described method for introducing a reprogramming factor, and in particular, the reprogramming factor and the CAR gene may be introduced simultaneously or sequentially depending on a desired time and conditions.

Specifically, as shown in blue of FIG. 2, the lentivirus expressing CAR may be transformed into PBMC at the time of conversion into iNK or into cells at the time of reprogramming induction process by inoculating into a culture medium selected from the steps of culturing in the CAR-iNK first medium, CAR-iNK second medium, or CAR-iNK third medium, simultaneously with the reprogramming factor or after introduction of the reprogramming factor. The method according to the present invention is significant in that CAR-expressing NK cells can be produced by introducing the CAR gene and the reprogramming factor into isolated cells other than NK cells. In addition, it is significant in that the time of introducing the CAR gene can be determined as desired by those skilled in the art during the reprogramming culturing process. Specifically, the time of introduction of the CAR gene, that is, the time of inoculation of the CAR-expressing lentivirus may be carried out simultaneously with the reprogramming factor (day 0), or at the step of culturing in the CAR-iNK second medium (day 12) or the CAR-iNK third medium (day 24) after introduction of the reprogramming factor, but is not limited thereto (FIGS. 2 and 6).

As used herein, the term “culture” means that the microorganism is grown under appropriately controlled environmental conditions. The culturing process of the present invention may be performed in a suitable culture medium and culture conditions known in the art. Such a culturing process may be easily adjusted for use by those skilled in the art according to the strain to be selected. For the purpose of the present invention, the culture is a process of converting cells into which reprogramming factors and/or CAR genes have been introduced into target cells of another lineage. The composition of the first medium, the second medium, or the third medium for culturing the cells into which the gene is introduced is a composition suitable for conversion into target cells, and for example, may include growth factors, cytokines, a GSK3β inhibitor, or an AHR antagonist, but is not limited thereto.

The first medium of a) may include a growth factor, cytokines, and a GSK3β inhibitor.

As used herein, the term “growth factor” means a polypeptide that promotes the division, growth, and differentiation of various cells. The growth factor may be, for example, EGF (epidermal growth factor), PDGF-AA (platelet-derived growth factor-AA), IGF-1 (insulin-like growth factor 1), TGF-β (transforming growth factor-β), FGF (fibroblast growth factors), SCF (stem cell factor), and FLT3 (FMS-like tyrosine kinase), and may specifically be any one or more selected from the group consisting of SCF, FLT3, and a combination thereof, but is not limited thereto.

As used herein, the “cytokines” are various proteins of relatively small size that are produced in cells and used for cell signaling, and can affect other cells, including themselves. They are generally known to be involved in the immune response to inflammation or infection. The cytokines may be, for example, IL (interleukin)-2, IL-3, IL-5, IL-6, IL-7, IL-11, IL-15, IL-21, IL-12, IL-18, BMP4 (bone morphogenetic protein 4), activin A, notch ligand, G-CSF (granulocyte-colony stimulating factor), SDF-1 (stromal cell-derived factor-1), etc., and may specifically be any one or more selected from the group consisting of IL-3, IL-6 IL-15, IL-7, IL-2, IL-21, IL-12, IL-18, and a combination thereof, but are not limited thereto.

For the purpose of the present invention, the growth factors and cytokines are included in the medium for directly reprogramming the isolated cells into the target cells, and the types of growth factors and cytokines are not particularly limited as long as they can be used for direct reprogramming.

As used herein, the term “GSK3β (glycogen synthase kinase 3 beta, glycogen synthase kinase-3β ) inhibitor” means a substance that suppresses or inhibits the activity of GSK3β. The GSK3β inhibitor may be, for example, 1-azakenpaullone, 2-D08, 3F8, 5-bromoindole, 6-Bio, A 1070722, aloisine A, AR-A014418, alsterpaullone, AZD-1080, AZD2858, bikinin, BIO, BIO-acetoxime, bisindolylmaleimide I, bisindolylmaleimide I hydrochloride, CAS 556813-39-9, cazpaullone, CHIR98014, CHIR98023, CHIR99021 (CT99021), CP21R7, dibromocantherelline, GSK-3β inhibitor I, VI, VII, X, XI, XV, GSK-3 inhibitor IX, XVI, hymenidin, hymenialdisine, HMK-32, I3M (indirubin-3-monoxime, indirubin, indole-3-acetamide, IM-12, kenpaullone, L803-mts, leucettine L41, lithium, lithium carbonate, LY-2090314, manzamine A MeBIO, meridianine A, NP00111, NP031115, NP031111, NSC 693868, palinurin, Ro 31-8220 methanesulfonate, SB-216763, SB-415286, TC-G 24, TCS 2002, TCS 21311, tideglusib, tricantin, trihydrochloride, tungstate, TWS-119, TZDZ-8, zinc, etc., and may specifically be CHIR99021, but is not limited thereto.

The first medium of a) may include SCF, FLT3, IL-3, and IL-6 and CHIR99021, but is not limited thereto.

The first medium of a) may further include any one or more selected from the group consisting of fetal bovine serum (FBS), antibiotics, and a combination thereof, but is not limited thereto.

The antibiotic may be penicillin/streptomycin, but is not limited thereto.

Specifically, the first medium of a) may include FBS, penicillin/streptomycin, SCF, FLT3, IL-3, and IL-6 and CHIR99021, but is not limited thereto.

More specifically, the first medium of a) may be StemSpan SFEM II containing 8% to 12% FBS, 0.1% to 2% penicillin/streptomycin, 80 ng/mL to 120 ng/mL human SCF, 80 ng/mL to 120 ng/mL human FLT3, 10 ng/mL to 30 ng/mL human IL-3, 10 ng/mL to 30 ng/mL human IL-6, and 2 μM to 7 μM CHIR99021, and more specifically StemSpan SFEM II containing 10% FBS, 1% penicillin/streptomycin, 100 ng/mL human SCF, 100 ng/mL human FLT3, 20 ng/mL human IL-3, 20 ng/mL human IL-6, and 5 μM CHIR99021, but is not limited thereto.

The second medium of b) may include a growth factor, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist.

The terms “growth factor” and “cytokine” are the same as described above.

As used herein, the term “AHR (aryl hydrocarbon receptor) antagonist” refers to a substance that down-regulates or reduces the activity of AHR, a ligand-activated transcription factor activated by TCDD (dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)). The AHR antagonist may be, for example, StemRegenin I (SRI; 4-(2-((2-benzo[b]thiphen-3-yl)-9-isopropyl-9H-purin-6-yl)amino)ethyl)phenol hydrochloride], CH-223191(1-methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide), etc., and specifically StemRegenin I, but is not limited as long as it plays a role in increasing the efficiency of direct reprogramming.

The second medium of b) may include SCF, FLT3, IL-15, IL-7, IL-2, and StemRegenin I, but is not limited thereto.

The second medium of b) may further include any one or more selected from the group consisting of FBS, antibiotics, and a combination thereof, but is not limited thereto.

The antibiotic may be penicillin/streptomycin, but is not limited thereto.

Specifically, the second medium of b) may include FBS, penicillin/streptomycin, SCF, FLT3, IL-15, IL-7, IL-2, and StemRegenin I, but is not limited thereto.

More specifically, the second medium of b) may include 8% to 12% FBS, 0.1% to 2% penicillin/streptomycin, 10 ng/mL to 30 ng/mL human SCF, 10 ng/mL to 30 ng/mL human FLT3, 100 IU/mL to 500 IU/mL human IL-2, 10 ng/mL to 30 ng/mL human IL-7, 10 ng/mL to 30 ng/mL human IL-15, and 1 μM to 3 μM StemRegenin I, and more specifically 10% FBS, 1% penicillin/streptomycin, 20 ng/mL human SCF, 20 ng/mL human FLT3, 200 IU/mL human IL-2, 20 ng/mL human IL-7, 20 ng/mL human IL-15, and 2 μM StemRegenin I, but is not limited thereto.

The third medium of c) may include cytokines, a GSK3β inhibitor, and an AHR antagonist.

The terms “cytokines”, “GSK3β inhibitor”, and “AHR antagonist” are the same as described above.

The third medium of c) may include IL-2, IL-15, IL-21, IL-12, IL-18, CHIR99021, and StemRegenin I, but is not limited thereto.

The third medium of c) may further include any one or more selected from the group consisting of FBS, antibiotics, and a combination thereof, but is not limited thereto.

The antibiotic may be penicillin/streptomycin, but is not limited thereto.

Specifically, the third medium of c) may include FBS, penicillin/streptomycin, IL-2, IL-15, IL-21, IL-12, IL-18, CHIR99021, and StemRegenin I, but is not limited thereto.

More specifically, the third medium of c) may be RPMI 1640 containing 8% to 12% FBS, 0.1% to 2% penicillin/streptomycin, 100 IU/mL to 500 IU/mL human IL-2, 40 ng/mL to 60 ng/mL human IL-12, 10 ng/mL to 30 ng/mL human IL-15, 90 ng/mL to 110 ng/mL human IL-18, 10 ng/mL to 30 ng/mL human IL-21, 1 μM to 3 μM CHIR99021, and 1 μM to 3 μM StemRegenin I, and more specifically RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin, 200 IU/mL human IL-2, 50 ng/mL human IL-12, 20 ng/mL human IL-15, 100 ng/mL human IL-18, 20 ng/mL human IL-21, 2 μM CHIR99021, and 2 μM StemRegenin I, but is not limited thereto.

In the method above, the isolated cells into which the reprogramming factor and the CAR gene are introduced may be cultured in the first medium of a) for 4 to 7 days, and in the second medium of b) for 10 to 14 days, and then in the third medium of c) for 14 days or more, but is not limited thereto.

In one embodiment of the present invention, the cells were cultured for different culture periods (3, 5, 7, and 9 days) in the CAR-iNK first medium, and then cultured in the CAR-iNK second medium and the third medium under the same conditions as in Example 1, and the CAR-iNK production yield was analyzed by comparison. As a result, it was confirmed that the highest CAR-iNK production yield of about 80% was obtained when the cells were cultured in the CAR-iNK first medium for 5 days (FIG. 7A).

In another embodiment of the present invention, it was confirmed that the CAR-iNK yield efficiency was increased when the cells were cultured in the CAR-iNK third medium containing 20 ng/mL human IL-21, 2 μM CHIR99021, 2 μM StemRegenin I (SR1), 50 ng/mL human IL-12, and 100 ng/mL human IL-18 (FIG. 7B). In addition, when the cells were cultured for 7 days or 14 days in the CAR-iNK third medium for each composition, it was confirmed that the CAR-iNK yield efficiency was increased up to 15 times when cultured for 14 days (FIG. 7B). This suggested that CAR-iNK cells exhibited the highest production efficiency (CD19-CAR-iNK cells: maximum 45.9% (FIG. 6A), MSLN-CAR-iNK cells: 43.7% (FIG. 4)), HER2-CAR-iNK cells: 47.8% (FIG. 5) when cultured in the CAR-iNK first medium for 5 days, in the CAR-iNK second medium for 12 days, and in the CAR-iNK third medium for 2 to 3 weeks.

The CAR-iNK cells produced according to the method of the present invention may express any one or more selected from the group consisting of CD56+, CD16+, CD3−, and a combination thereof, but is not limited thereto.

The “CD56+”, “CD16+”, and “CD3−” are indicators on the surface of NK cells, and in the present invention, the expression of CD56+, CD16+, and CD3− was analyzed through flow cytometry to determine whether CAR-iNK cells were produced (FIGS. 3 to 6).

In addition, the CAR-iNK cells produced according to the method of the present invention may have an excellent killing ability against various cancer cells.

In one embodiment of the present invention, the frequency (%) of CD107a+ cells and interferon-gamma+cells having an cancer cell lysis ability was increased in cancer cells co-cultured with the CAR-iNK cells (FIGS. 8 to 15), and as a result of confirming the killing ability of iNK cells against blood cancer cell lines, pancreatic cancer cell lines, prostate cancer cell lines, colorectal cancer cell lines, lung cancer cell lines, liver cancer cell lines, gastric cancer cell lines, and melanoma cell lines, it was confirmed that they exhibited an excellent killing ability (FIGS. 16 to 18). Additionally, it was confirmed that the tumor size was significantly reduced on the 14th day after the injection of CAR-iNK cells in a mouse model with pancreatic cancer, compared to the control group which was not injected with iNK cells or iNK cells into which the CAR gene was not introduced (FIG. 19).

Another aspect of the present invention provides CAR-iNK cells produced according to the method above. The terms used herein are the same as described above.

As described above, the CAR-iNK cells may have an excellent killing ability against various cancer cells.

Still another aspect of the present invention provides a cell therapy composition for preventing or treating cancer, including the CAR-iNK cells produced according to the method above, as an active ingredient.

The terms used herein are the same as described above.

As used herein, the term “prevention” refers to all actions that suppress or delay cancer by the administration of the composition.

As used herein, the term “treatment” refers to all actions that alleviate or beneficially change the symptoms of cancer by the administration of the composition.

As used herein, the term “cell therapeutic agent” refers to a drug for treatment, diagnosis, and prevention (U.S. FDA guidance) containing cells or tissues prepared from humans via isolation, culture, and specialized manipulations, and to a drug for treatment, diagnosis, and prevention prepared by any process including proliferating and selecting autologous, homologous, or heterologous cells ex vivo, or modifying the biological characteristics of cells, so as to restore the function of cells or tissues.

The cell therapy composition may have an efficacy of preventing or treating cancer by including the CAR-iNK cells produced according to the method of the present invention.

The cell therapy composition may contain the CAR-iNK cells at 1.0×10⁴ cells/mL to 1.0×10¹⁰ cells/mL, preferably 1.0×10⁵ cells/mL to 1.0×10⁹ cells/mL, based on the total weight of the composition, but is not limited thereto.

The cell therapy composition may be administered by formulating it into a pharmaceutical formulation in the form of unit dosage suitable for administration to the body of a patient by conventional methods in the pharmaceutical field, and it contains an effective amount by a single dose or in divided doses. For this purpose, a formulation for parenteral administration may preferably include injection formulation such as an injection ampoule, infusion formulation such as an infusion bag, and spray formulation such as an aerosol, etc. The injection ampoule may be mixed with injection solution such as saline solution, glucose, mannitol, and Ringer's solution just before use. Further, the cells can be carried by an infusion bag textured with polyvinyl chloride or polyethylene, and examples thereof may include infusion bags manufactured by Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo.

The pharmaceutical formulation may additionally include one or more pharmaceutically acceptable inactive carriers in addition to the active ingredient, for example, a preservative, analgesic controller, solubilizer, or stabilizer for injection formulation, and a base, excipient, lubricant, or preservative for topical formulation.

The thus-produced cell therapy composition of the present invention or a pharmaceutical formulation thereof may be administered in accordance with any conventional method in the art together with other cells used for treatment of cancer, or in the form of a mixture therewith. Direct engraftment or transplantation to the diseased area of a patient in need of treatment, or direct transplantation or injection into the abdominal cavity is preferred, but the method is not limited thereto. Further, both non-surgical administration using a catheter and surgical administration such as injection or transplantation after incision of the diseased area are possible. In addition, the composition may also be administered parenterally by the conventional method, for example, transplantation of cells into the hematopoietic system, in addition to direct administration to the lesion.

The cell therapy composition of the present invention may be administered in an amount ranging from about 0.0001 mg/kg to 1000 mg/kg, preferably 0.001 mg/kg to 100 mg/kg once per day or in several divided doses per day. However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the disease to be treated, the severity of the disease, the route of administration, and the body weight, age and sex of a patient, and therefore, the above dose should not be intended to limit the scope of the present invention in any way.

Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, including the CAR-iNK cells produced according to the method above, as an active ingredient.

The terms used herein are the same as described above.

In the present invention, the cancer may be a cancer showing prevention or treatment results due to an immune response of CAR-iNK cells, etc. The cancer may be, for example fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma cancer, osteogenic sarcoma, myeloma, myeloma, myelodysplasia, lymphoma, non-Hodgkin's lymphoma, blood cancer, melanoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial sarcoma, mesothelioma, Ewing's sarcoma, gastric cancer, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colon cancer, colorectal cancer, rectal cancer, pancreatic cholangiocarcinoma, pancreatic cancer, biliary tract cancer, gallbladder cancer, liver cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, preleukemia, leukemia, acute leukemia, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoblastic leukemia (ALL); chronic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous adenocarcinoma, papillary thyroid cancer, cyst cancer, medullary thyroid cancer, bronchogenic carcinoma, renal cell carcinoma, liver cancer, biliary duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, head and neck cancer, brain cancer, glioma, astrocytoma, renal cell carcinoma, glioblastoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, and sarcoma, and may specifically be a cancer associated with expression of any one or more of CD19, MSLN, or HER2, for example, any one or more selected from the group consisting of myelodysplasia, myelodysplastic syndrome, preleukemia, blood cancer, acute leukemia, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoblastic leukemia (ALL), chronic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, lymphoma, myeloma, pancreatic cancer, biliary tract cancer, lung cancer, ovarian cancer, breast cancer, uterine cancer, rectal cancer, colorectal cancer, colon cancer, bone marrow cancer, liver cancer, brain cancer, prostate cancer, stomach cancer, glioma, melanoma, squamous cell carcinoma, head and neck cancer, renal cell cancer, glioblastoma, medulloblastoma, sarcoma, and a combination thereof, and more specifically any one or more selected from the group consisting of blood cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, liver cancer, stomach cancer, melanoma, and a combination thereof, but is not limited thereto.

The pharmaceutical composition may have an efficacy of preventing or treating cancer by including the CAR-iNK cells produced according to the method of the present invention.

The pharmaceutical composition of the present invention may contain the CAR-iNK cells at 1.0×10⁴ cells/mL to 1.0×10¹⁰ cells/mL, preferably 1.0×10⁵ cells/mL to 1.0×10⁹ cells/mL, based on the total weight of the composition, but is not limited thereto.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical compositions, and the carrier may include a carrier which does not occur naturally. The carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and minerals.

Further, the pharmaceutical composition may be formulated according to a conventional method into a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution for internal use, an emulsion, a syrup, a sterilized aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized preparation, a transdermal preparation, a gel, a lotion, an ointment, a cream, a patch, a cataplasma form, a paste, a spray, a skin emulsion, a skin suspension, a transdermal patch, a drug-containing bandage, or a suppository for use.

Specifically, the preparation may be formulated with commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid formulations for oral administration may include tablets, pills, powders, granules, capsules, etc., but are not limited thereto. Such solid formulations may be prepared by mixing with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate or talc may also be used. Liquid formulations for oral administration may be prepared by adding various excipients, for example, wetting agents, flavoring agents, aromatics, preservatives, etc. in addition to liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. The non-aqueous solutions and the suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyloleate, etc. The base for suppositories may include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount which is sufficient to treat diseases at a reasonable benefit/risk ratio applicable to any medical treatment. The effective dosage level may be determined depending on factors including a kind of a subject and severity, age, sex, activity of a drug, drug sensitivity, administration time, administration route, excretion rate, duration of treatment, drugs used concurrently, and other factors known in the medical field. For example, the pharmaceutical composition may be administered in a daily dosage of 0.0001 mg/kg to 1000 mg/kg, and specifically 0.001 mg/kg to 100 mg/kg, and the dose may be administered once per day or in several divided doses per day.

The pharmaceutical composition may be administered alone as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with existing therapeutic agents. The composition may be administered in a single or multiple dosage form. It is important to administer the composition in a minimum amount that may exhibit a maximum effect without causing side effects, considering all of the above-described factors. The amount may be readily determined by those skilled in the art.

As used herein, the term “administration” means introducing the composition of the present invention into a subject by any suitable method. The administration route of the composition may be administered through any general route as long as it can reach the target tissue, including intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, and intranasal administration, but is not limited thereto.

As used herein, the term “subject” refers to any animal, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs having cancer or at risk of having cancer. As long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention into a subject, any type of subject may be included without limitation.

Even another aspect of the present invention provides a method for treating cancer, including administering the cell therapy composition or the pharmaceutical composition to a subject except a human.

The terms used herein are the same as described above.

The present invention provides a simplified production process because CAR-iNK cells are produced from isolated cells through direct reprogramming and a production time of at least 32 days, which is shorter than that obtained by the method in which the CAR gene was introduced after producing NK cells using conventional reprogramming techniques, thereby reducing costs, has excellent NK cell production efficiency of 47.8% at maximum, and ensures safety as the cells are produced without passing through pluripotent stem cells, thereby showing an excellent CAR-iNK cell production effect that is distinguished from that of the conventional reprogramming techniques.

In addition, the present invention is distinguished from the conventional method of obtaining NK cells through direct harvesting, stem cell differentiation, etc. in that NK cells can be directly produced by culturing cells, which have a different cell lineage from NK cells and are easy to collect, in a reprogramming medium, and thus, the present invention is meaningful in that it can provide a wide range of options for cell types and quality.

Further, the CAR-iNK cells produced according to the method of the present invention have an excellent cancer cell killing ability as described above, and thus can be provided as a cell therapy composition and a pharmaceutical composition for preventing or treating cancer including the same.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the constitution and effect of the present invention will be described by way of specific Examples. However, these Examples are provided for illustrative purposes only, and the scope of the invention is not intended to be limited by these Examples.

Example 1 Construction of Lentiviral Vectors Encoding CAR

In order to construct double cistron lentiviral vectors encoding CAR (chimeric antigen receptor), four CAR genes each binding to CD19, HER2, (human epidermal growth factor receptor 2) or MSLN (mesothelin) were constructed. Each of the above CAR genes was constructed to include genes encoding an extracellular domain, a transmembrane domain, and an intracellular domain including an antibody domain (scFv). Specifically, each of the CAR genes was constructed as follows (FIG. 1):

i) CD19-CAR1 gene including CD8 leader (SEQ ID NO: 1), CD19 scFv (SEQ ID NO: 2), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane (TM) domain (SEQ ID NO: 6), Fc-γ (gamma) receptor (SEQ ID NO: 7), and GFP (green fluorescent protein) (SEQ ID NO: 11);

ii) CD19-CAR2 gene including CD8 leader (SEQ ID NO: 1), CD19 scFv (SEQ ID NO: 2), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane domain (SEQ ID NO: 6), CD28 intracellular domain (SEQ ID NO: 8), CD3 (zeta) (SEQ ID NO: 9), IRES (internal ribosome entry site) (SEQ ID NO: 10), and GFP (SEQ ID NO: 11);

iii) MSLN-CAR gene including CD8 leader (SEQ ID NO: 1), MSLN (mesothelin) scFv (SEQ ID NO: 3), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane domain (SEQ ID NO: 6), CD28 intracellular domain (SEQ ID NO: 8), CD3 (SEQ ID NO: 9), IRES (SEQ ID NO: 10), and GFP (SEQ ID NO: 11); and

iv) HER2-CAR gene including CD8 leader (SEQ ID NO: 1), HER2 (human epidermal growth factor receptor 2) scFv (SEQ ID NO: 4), CD8 hinge (SEQ ID NO: 5), CD8 transmembrane domain (SEQ ID NO: 6), CD28 intracellular domain (SEQ ID NO: 8), CD3 (SEQ ID NO: 9), IRES (SEQ ID NO: 10), and GFP (SEQ ID NO: 11).

The IRES was inserted for cloning the CAR gene into a vector constituting a double cistron. For each vector containing the four CAR genes, each domain of FIG. 1 was synthesized from CAR1 (Addgene ID: 113014), and CAR-expressing lentiviruses were constructed through overlap PCR (Gibson assembly).

Example 2 Direct Reprogramming of NK Cells from PBMC

Isolated peripheral blood mononuclear cells (PBMC) were cultured for 4 days in the culture solution (Stempro SFEM II containing 2.5% Stem Pro-34 nutrient supplement, 2 mM Glutamax I, 1% penicillin/streptomycin, 20 ng/mL human IL (interleukin)-3, 20 ng/mL human IL-6, 100 ng/mL human stem cell factor (SCF), and 100 ng/mL human FLT3 (FMS-like tyrosine kinase)), while changing the medium once every 2 days.

In order to transform the reprogramming factors (Oct4, Sox2, Klf4, and Myc) into the PBMC, the Sendai virus system [Oct4, Sox2, Klf4, and Myc-expressing RNA-based Sendai virus (CytoTune 2.0 Sendai Reprogramming Kit, Thermo Scientific); OSKM-SeV] expressing the reprogramming factors and the 4 types of lentiviruses expressing the 4 types of CARs of Example 1 were used. Specifically, in order to transform PBMC with the reprogramming factors, the cells were cultured in a standard culture medium (SCM medium) containing the Sendai virus (5 MOI), PBMC and polybrene (4 μg/mL) for 1 day, and then sequentially cultured in the first medium, second medium, and third medium shown below. The cells transformed by the method (2×10⁵ cells/48-well plate) were cultured in the first CAR-iNK medium (StemSpan SFEM II containing 10% FBS, 1% penicillin/streptomycin, 100 ng/mL human SCF, 100 ng/mL human FLT3, 20 ng/mL human IL-3, 20 ng/mL human IL-6, and 5 μM CHIR99021) including the GSK3β (glycogen synthase kinase 3β) inhibitor for 5 to 6 days, and then cultured in the CAR-iNK second medium (StemSpan SFEM II containing 10% FBS, 1% penicillin/streptomycin, 20 ng/mL human SCF, 20 ng/mL human FLT3, 200 IU/mL human IL-2, 20 ng/mL human IL-7, 20 ng/mL human IL-15, and 2 μM StemRegenin I) including the AHR (aryl hydrocarbon receptor) antagonist for 12 days, and subsequently in the CAR-iNK third medium (RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin, 200 IU/mL human IL-2, 50 ng/mL human IL-12, 20 ng/mL human IL-15, 100 ng/mL human IL-18, 20 ng/mL human IL-21, 2 μM CHIR99021, and 2 μM StemRegenin I) including the GSK3β inhibitor and AHR antagonist for 14 days or more to induce the cells into NK cells (FIG. 2). The CAR-expressing lentiviruses were inoculated into a culture medium selected from the step of culturing in the CAR-iNK first medium (day 4), the CAR-iNK second medium (day 14), or the CAR-iNK third medium (day 24) simultaneously with the reprogramming factor (day 0) or after introduction of the reprogramming factor, as shown in FIG. 2, and thus were transformed into the cells during the reprograming process by which PBMC were converted into NK cells.

In order to confirm whether NK cells introduced with the CD19-CAR1 gene, CD19-CAR2 gene, MSLN-CAR gene, or HER2-CAR gene were prepared through the direct reprogramming, the cells were stained with the CD56 antibody, CD3 antibody, CD19 antigen, MSLN antigen, and HER2 antigen, and then iNK cell group (CD56+ and CD3−), CD19 CAR-iNK cell group (CD56+ and CD19+), MSLN CAR-iNK cell group (CD56+and MSLN+), and HER2 CAR-iNK cell group (CD56+and HER2+) were analyzed using flow cytometry. Specifically, NK (iNK) cells induced through direct reprogramming were added to a phosphate buffer (FACS buffer) containing 1% BSA (bovine serum albumin) and 2 mM EDTA (ethylenediaminetetraacetic acid), supplemented with fluorescent-labeled antibodies and antigens, and reacted at room temperature for 20 minutes, and subsequently, the cells were washed and recovered using a centrifuge, and then analyzed by FACS (BD Bioscience).

In addition, in order to confirm whether each CAR gene was inserted into the genome of iNK cells, the genomic DNA of each cell was analyzed with primers (SEQ ID NOS: 12 and 13) for CD19 CAR, primers (SEQ ID NOS: 14 and 15) for MSLN CAR, and primers (SEQ ID NOS: 16 and 17) for HER2 CAR through PCR gene analysis.

As a result, it was confirmed that CD56+CD3−CD19-CAR-iNK cells were produced with a maximum efficiency of 45.9% (FIG. 3 and FIG. 6A), that CD56+CD3−MSLN-CAR-iNK were produced with a maximum efficiency of 43.7% (FIG. 4), and that CD56+CD3− HER2-CAR-iNK cells were produced with a maximum efficiency of 47.8% (FIG. 5).

Further, as shown in FIG. 6, when the CAR-expressing lentiviruses were inoculated into a culture medium selected from the step of culturing in the CAR-iNK second medium (day 14) or CAR-iNK third medium (day 24), simultaneously with the reprogramming factor (day 0) or after introduction of the reprogramming factor, it was confirmed that CD56+CD3−CD19-CAR-iNK cells were produced with an efficiency of 15.5%, 31.6%, and 45.9%.

Example 3: Optimization of Incubation Period

3-1. Optimization of Incubation Period in CAR-iNK First Medium

The cells were cultured in the CAR-iNK first medium for different incubation periods (3, 5, 7, and 9 days), and then cultured in the CAR-iNK second medium and the third medium under the same conditions as in Example 1, and the CAR-iNK production yield was analyzed by comparison.

As a result, it was confirmed that the highest CAR-iNK production yield of about 80% was obtained when cultured in the CAR-iNK first medium for 5 days (FIG. 7A).

3-2. Optimization of Composition of CAR-iNK Third Medium The CAR-iNK production yield was analyzed by comparison by culturing in the CAR-iNK first medium and the second medium under the same conditions as in Example 1, and then cultured in the CAR-iNK third medium having different compositions.

As a result, it was confirmed that the CAR-iNK yield efficiency was increased when cultured in the CAR-iNK third medium containing 20 ng/mL human IL-21, 2 μM CHIR99021, 2 μM StemRegenin 1 (SR1), 50 ng/mL human IL-12, and 100 ng/mL human IL-18 (FIG. 7B). In addition, it was confirmed that in the case of culturing the cells for 7 days or 14 days in the CAR-iNK third medium for each composition, the CAR-iNK yield efficiency was increased up to 15 times when cultured for 14 days (FIG. 7B).

Based on the above results, the CAR-iNK cells were cultured in the CAR-iNK first medium for 5 days, in the CAR-iNK second medium for 12 days, and in the CAR-iNK third medium for 2 to 3 weeks.

Example 4 Quantitative Analysis of CD107a+ Cells

In order to verify the cancer cell killing potential of the CAR-iNK cells produced in Example 2, the frequency of CD107a+cells having a cancer cell lysis ability, which are expressed after co-culturing CAR-iNK cells with cancer cells, was quantitatively analyzed. Specifically, 1 ×10⁶ cells/mL of Raji (Raji B, human B lymphocytes; Burkitt's lymphoma), Jurkat T (immortalized human T lymphocytes), SNU-291 (human B-lymphoblastoid cells), SNU-817 (human B-lymphoblastoid cells), HCT116 (human colon cancer cells), NIC-H460 (human lung cancer cells), HepG2 (human liver hepatocellular carcinoma cells), Mia-paca-2 (human pancreas ductal adenocarcinoma cells), PC3 (PC-3, human prostate cancer cells), which are cancer cells, and 1×10⁶ cells/mL of CAR-iNK cells were each dispensed in a 6-well plate in an amount of 1 mL, and centrifuged at 400 g for 1 minute, followed by culturing in a cell incubator at 37° C. in the presence of 5% CO₂ for 2 hours or 16 hours, and then the frequency of CD107a+ cells was confirmed through flow cytometry. Specifically, the frequency of CD107a+ cells was determined through FACS analysis after reacting the CAR-iNK cells in FACS buffer supplemented with fluorescent-labeled antibodies against CD56 and CD107a at room temperature for 20 minutes, and then washing and recovering the cells using a centrifuge.

As a result, when the cancer cells were co-cultured with CD19-CAR-iNK cells (FIGS. 8 and 9), MSLN-CAR-iNK cells (FIG. 10), and HER2-CAR-iNK cells (FIG. 11), it was confirmed that the frequency (%) of CD107a+iNK cells was increased, compared to the iNK control group into which the CAR gene was not introduced or the control group (−) without co-cultivation.

Example 5 Quantitative Analysis of IFN-gamma-Expressing Cells

In order to verify the cancer cell killing potential of the CAR-iNK cells produced in Example 2, the frequency of IFN-gamma cells, which are expressed after co-culturing CAR-iNK cells with cancer cells, was quantitatively analyzed. Specifically, 1 ×10⁶ cells/mL of Raji, SNU-291, SNU-817, HepG2, NIC-H460 (human lung cancer cells), KATO3 (KATOIII, human gastric cancer cells), CFPAC-1 (human ductal pancreatic adenocarcinoma cells), and 1×10⁶ cells/mL of CAR-iNK cells were each dispensed in a 6-well plate in an amount of 1 mL, and centrifuged at 400 g for 1 minute, followed by culturing in a cell incubator at 37° C. in the presence of 5% CO₂ for 2 hours or 16 hours, and then the frequency of IFN-gamma cells was confirmed through flow cytometry. Specifically, the frequency of IFN-gamma cells was determined after reacting the CAR-iNK cells in a phosphate buffer containing 0.5% Tween 20 and 0.5% BSA at room temperature for 20 minutes, and then washing and recovering the cells using a centrifuge. The recovered cells were reacted at room temperature for 20 minutes in FACS buffer supplemented with fluorescent-labeled antibodies against CD56 and IFN-gamma, and then the cells were washed and recovered using a centrifuge and subjected to FACS analysis.

As a result, it was confirmed that the frequency (%) of IFN-gamma+iNK cells was increased when the cancer cells were co-cultured with CD19-CAR-iNK cells (FIGS. 12 and 13), MSLN-CAR-iNK cells (FIG. 14), and HER2-CAR-iNK cells (FIG. 15), compared to the iNK control group into which the CAR gene was not introduced or the control group (−) without co-cultivation.

Example 6 Measurement of Cancer Cell Killing Ability of CAR-iNK Cells

In order to measure the cancer cell killing ability of the CAR-iNK cells produced in Example 1, the cell killing ability was measured using calcein-AM. Specifically, Raji, SNU-291, SNU-817, Mia-paca-2, CFPAC-1, PC3 (PC-3), LNcap (androgen-sensitive human prostate adenocarcinoma cells), DU145 (human prostate cancer cells), HCT116, A549 (human lung carcinoma cells), NIC-H460, HepG2, KATO3 (KATOIII), SK-MEL-3 (human melanoma cells), which are cancer cells, were diluted to 1×10⁵ cells/mL in DMEM medium containing 10% fetal bovine serum, added with calcein-AM to a concentration of 25 μM, and washed with DMEM medium after culturing for 1 hour at 37° C., and accordingly, calcein-labeled target cancer cells were prepared.

The iNK cells were prepared by diluting the cells to a density of 0.25×10⁵ cells/mL and 1 ×10⁵ cells/mL using a culture solution, and then dispended in a 96-well plate in an amount of 100 mL. The thus-prepared calcein-labeled target cancer cells (1×10⁵ cells/mL) were added to a 96-well plate in an amount of 100 pUwell, centrifuged at 400 g for 1 minute, and then cultured in a cell incubator at 37° C. for 4 hours in the presence of 5% CO₂, and subsequently, 100 μL of the supernatant was taken from each well and measured with a fluorescence plater reader (485 nm/535 nm). The cell killing ability (%) was calculated according to the following formula.

Cancer Cell Killing Ability(%)={(Measured ValueΔMinimum Value)/(Measured Value−Minimum Value)}×100

In the above formula, the minimum value is the measured value of a well in which only calcein-labeled target cancer cells exist, and the maximum value is the measured value of a well in which cells are completely lysed by adding 0.1% TritonX-100 to the calcein-labeled target cancer cells.

As a result, it was confirmed that CD19-CAR-iNK cells (FIG. 16), MSLN-CAR-iNK cells (FIG. 17), and HER2-CAR-iNK cells (FIG. 18) exhibited a high cancer cell killing ability, compared to iNK control group, and that the cancer cell killing ability was increased in proportion to the number of iNK cells.

Example 7 Verification of In Vivo Cancer Cell Killing Ability of CAR-iNK Cells

200 μL (1×10⁷ cells/mL) of CFPAC-1 expressing luciferase were subcutaneously injected into the back of 8-week-old nude mice (Balb/c-nude mice, average weight 20-25 g) to prepare a pancreatic cancer cell xenograft mouse animal model. The next day, after injecting 200 μL of PBS as a negative control, or iNK and MSLN-CAR-iNK cells as an experimental group at the same dose (1 ×10⁷ cells/mL PBS), 150 μL of D-luciferin dissolved in PBS was injected intraperitoneally at 7-day intervals (150 μg/mL, Promega), and the tumor size was confirmed through IVIS 100 (PerkinElmer) after 15 to 20 minutes.

As a result, it was confirmed that the tumor size formed in the iNK (5.35×10⁹ radiance) and CAR-NK (3.83×10⁹ radiance) cell experimental groups was significantly reduced compared to the tumor size (1.21 ×10¹⁰ radiance) of the control group formed under the condition of PBS injection on the 14th day, thereby confirming that the CAR-iNK cells exhibited an excellent anticancer effect compared to iNK cells (FIG. 19).

From the results of the above Examples, it was confirmed that the CAR-iNK cells having an excellent anticancer ability can be prepared by directly reprogramming the isolated cells.

Those of ordinary skill in the art will recognize that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within the scope of the present invention. 

1. A method for producing CAR-iNK (induced natural killer) cells, comprising: culturing isolated cells, into which a reprogramming factor and a CAR (chimeric antigen receptor) gene are introduced, sequentially in a) a first medium containing growth factors, cytokines, and a GSK3β (glycogen synthase kinase 3 beta) inhibitor; b) a second medium containing growth factors, cytokines, and an AHR (aryl hydrocarbon receptor) antagonist; and c) a third medium containing cytokines, a GSK3β inhibitor, and an AHR antagonist, and thereby directly reprogramming into NK cells.
 2. The method of claim 1, wherein the CAR gene is introduced into the isolated cells during any one selected from culturing in the first medium of a), the second medium of b), or the third medium of c), when introducing the reprogramming factor.
 3. The method of claim 1, wherein the growth factor is any one or more selected from the group consisting of SCF (stem cell factor), FLT3 (FMS-like tyrosine kinase), and a combination thereof.
 4. The method of claim 1, wherein the cytokine is any one or more selected from the group consisting of IL (interleukin)-3, IL-6 IL-15, IL-7, IL-2, IL-21, IL-12, IL-18, and a combination thereof.
 5. The method of claim 1, wherein the first medium of a) contains SCF, FLT3, IL-3, IL-6, and CHIR99021.
 6. The method of claim 1, wherein the second medium of b) contains SCF, FLT3, IL-15, IL-7, IL-2, and StemRegenin I.
 7. The method of claim 1, wherein the third medium of c) contains IL-2, IL-15, IL-21, IL-12, IL-18, CHIR99021, and StemRegenin I.
 8. (canceled)
 9. The method of claim 1, wherein the isolated cells, into which a reprogramming factor and a CAR gene are introduced, are cultured in the first medium of a) for 4 to 7 days, in the second medium of b) for 10 to 14 days, and then in the third medium of c) for 14 days or more.
 10. The method of claim 1, wherein the reprogramming factor is any one or more selected from the group consisting of Lin28, Asc11, Pitx3, Nurr1, Lmx1a, Nanog, Oct4, Oct3, Sox2, Klf4, Myc, and a combination thereof.
 11. The method of claim 10, wherein the reprogramming factor is Oct4, Sox2, Klf4, and Myc.
 12. The method of claim 1, wherein the CAR gene is any one or more selected from the group consisting of: i) a CAR gene comprising CD8 leader, CD19 scFv, CD8 hinge, CD8 transmembrane domain, and an Fc-γ receptor; ii) a CAR gene comprising CD8 leader, CD19 scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3ζ, and IRES (internal ribosome entry site); iii) a CAR gene comprising CD8 leader, MSLN (mesothelin) scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3ζ, and IRE; and iv) a CAR gene comprising CD8 leader, HER2 (human epidermal growth factor receptor 2) scFv, CD8 hinge, CD8 transmembrane domain, CD28 intracellular domain, CD3ζ, and IRES.
 13. The method of claim 12, wherein the CAR gene further comprises GFP (green fluorescent protein).
 14. The method of claim 1, wherein the isolated cells, into which a reprogramming factor and a CAR gene are introduced, are somatic cells except NK cells.
 15. The method of claim 1, wherein the CAR-iNK cells produced above express any one or more selected from the group consisting of CD56+, CD16+, CD3−, and a combination thereof.
 16. CAR-iNK cells produced according to the method of claim
 1. 17. A cell therapy composition for preventing or treating cancer, comprising the CAR-iNK cells produced according to the method of claim 1, as an active ingredient.
 18. A pharmaceutical composition for preventing or treating cancer, comprising the CAR-iNK cells produced according to the method of claim 1, as an active ingredient.
 19. The pharmaceutical composition of claim 18, wherein the cancer is associated with the expression of any one or more selected from the group consisting of CD19, MSLN, or HER2.
 20. The pharmaceutical composition of claim 19, wherein the cancer which is associated with the expression of any one or more selected from the group consisting of CD19, MSLN, or HER2 is any one or more selected from the group consisting of myelodysplasia, myelodysplastic syndrome, preleukemia, blood cancer, acute leukemia, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoblastic leukemia (ALL), chronic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, lymphoma, myeloma, pancreatic cancer, biliary tract cancer, lung cancer, ovarian cancer, breast cancer, uterine cancer, rectal cancer, colorectal cancer, colon cancer, bone marrow cancer, liver cancer, brain cancer, prostate cancer, stomach cancer, glioma, melanoma, squamous cell carcinoma, head and neck cancer, renal cell cancer, glioblastoma, medulloblastoma, sarcoma, and a combination thereof.
 21. The pharmaceutical composition of claim 20, wherein the cancer which is associated with the expression of any one or more selected from the group consisting of CD19, MSLN, or HER2 is any one or more selected from the group consisting of blood cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, liver cancer, stomach cancer, and melanoma. 